The major aim of the grant is to understand the roles of C/EBP-like transcription factors in the regulation of liver-specific genes using in vitro cell culture model systems and null mutations produced in transgenic animals by homologous recombination strategies. We have characterized an in vitro model of the inflammatory or acute phase response in man using a human hepatoma cell line, Hep 3B2. A subset of the acute phase response genes are transcriptionally regulated by DNA binding proteins of the C/EBP family. Seven family members have been identified that utilize a common consensus element. However, the role of each of these gene products in liver-specific gene regulation during the acute phase and during differentiation is not well understood. We will use null mutants of the Hep 3B2 cells generated by antisense methodologies or homologous recombination to clarify the roles of the individual members of the family. We will clone the full complement of C/EBP-like genes expressed in hepatoma cells and in liver. We will characterize the promoter elements of the C/EBPalpha gene and define cis-elements that influence C/EBPalpha expression. Cis-elements which are regulated by other C/EBP family members will be sought and characterized in detail. Finally, we will examine the role of C/EBPalpha in development and in the acute phase response of mice. For these studies, we will introduce null mutations by homologous recombination in embryonic stem cells to generate animals which are mutant for one or the other of these loci.